Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 30 December 2009 and 23 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source:
Harlan (UK) Ltd.

- Age at study initiation:
eight to twelve weeks old

- Weight at study initiation:
200g to 350g.

- Fasting period before study:
Not applicable

- Housing:
solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK).

- Diet:
ad libitum

- Water:
ad libitum

- Acclimation period:
at least five days


ENVIRONMENTAL CONDITIONS

- Temperature:
19 - 25°C

- Humidity:
30 - 70%

- Air changes (per hr):
at least fifteen changes per hour

- Photoperiod (hrs dark / hrs light):
twelve hours continuous light and twelve hours darkness


IN-LIFE DATES:
From: Day 0 To: Day 14

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
The test item was vaporised by injecting it directly into the air supply to the exposure chamber. The test item was contained in a glass syringe located on an infusion pump thus providing a constant supply of test item into the air stream. Immediately after the injection site, the air supply was ducted, via suitable tubing and a conical flask, through a water bath, maintained at approximately 50°C, to ensure complete vaporisation.

The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure. A schematic diagramof the dynamic (continuous flow) system employed please see "Illustration picture graph" section.

- Exposure chamber volume:
approximately 30 litres(dimensions: 28 cm diameter x 50 cm high)

- Method of holding animals in test chamber:
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.

- Source and rate of air:
Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the test item. The chamber flow rate was maintained at 50 L/min providing 100 air changes per hour.

- Method of conditioning air:
water trap and respiratory quality filters

- System of generating particulates:
The test item was vaporised by injecting it directly into the air supply to the exposure chamber. The test item was contained in a glass syringe located on an infusion pump thus providing a constant supply of test item into the air stream. Immediately after the injection site, the air supply was ducted, via suitable tubing and a conical flask, through a water bath, maintained at approximately 50°C, to ensure complete vaporisation.

- Method of particle size determination:
Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).

- Treatment of exhaust air:
filtered

- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. Individual values are given in Appendix 7 in Any other information on results incl, tables section.

-Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowborough,) located in a sampling port in the animals breathing zone during each exposure period. The test atmosphere was generated to contain at least 19% oxygen. Individual values are given in Appendix 8 in Any other information on results incl, tables section.

TEST ATMOSPHERE
- Brief description of analytical method used:
During the study, each sample consisted of 2 litres of test atmosphere drawn through an impinger containing acetone (made up to 80ml). The concentration of Reaction Products of C4 alcohols and C4 alkenes in the impinger was determined by GC using an external standard technique.

- Samples taken from breathing zone:
yes


VEHICLE
- Composition of vehicle (if applicable):
Not applicable

- Concentration of test material in vehicle:
Not applicable

- Justification of choice of vehicle:
Not applicable

- Lot/batch no. (if required):
Not applicable

- Purity: Not applicable


TEST ATMOSPHERE (if not tabulated)
Not applicable


CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Not applicable.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
A GC method of analysis was developed and used to measure the chamber atmosphere concentrations of Reaction Products of C4 alcohols and C4 alkenes in an acute inhalation study.
Duration of exposure:
4 h
Concentrations:
Mean Achieved (mg/L): 18.3
Standard Deviation: 1.36
Nominal (mg/L):20.7


No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for fourteen days. Individual body weights were recorded prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs.
Statistics:
Data evaluations included the relationship, if any, between the animals’ exposure to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, necropsy findings, bodyweight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test material was made.

Results and discussion

Preliminary study:
Not applicable
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 18.3 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: CL not given
Mortality:
No deaths occurred
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a
Body weight:
All animals exhibited a slight bodyweight loss during Days 0 to 1 post-exposure, with the exception of one female animal which exhibited a further bodyweight loss during the second week post-exposure, normal bodyweight development was noted during the study.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.
Other findings:
Not applicable.

Any other information on results incl. tables

Exposure Chamber Atmosphere Concentration

Prior to the start of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fibre filters and recording their weights. The filters were then dried in a desiccator between 19 and 20°C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The mean non-volatile component of the batch used during the study was found to be 0.00% (n=10). It was therefore considered that a vapour study should be conducted and chemical analysis should be employed in order to determine test atmosphere concentrations.

The test atmosphere was sampled five times during the exposure period. The sampling procedure involved 2 litres of test atmosphere being drawn through an impinger containing acetone (made up to 80ml). The samples were then submitted for chemical analysis. The method of analysis is given in attatched Appendix 10.

The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber. The nominal concentration was similar to the actual concentration showing the ease at which atmospheres could be generated from this test item.

Appendix 1      Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

18

2

50

20.0

62

2

50

18.4

124

2

50

17.0

182

2

50

19.2

232

2

50

16.9

 

Mean achieved atmosphere concentration (mg/L) =18.3

Standard deviation =1.36

Nominal concentration:

TestItemused (g)

251

Air Flow (L/min)

50

Total Generation Time (mins)

243

Nominal Concentration (mg/L)

20.7

Appendix 7      Temperature and Relative Humidity in Exposure Chamber

Time

(Minutes)

Chamber Temperature (ºC)

During Exposure

Chamber Relative Humidity (%) During Exposure

0

21

60

30

21

55

60

22

56

90

22

54

120

22

54

150

22

54

180

22

54

210

22

55

240

22

54

 


Appendix 8      Air Flow and Oxygen Concentration in Exposure Chamber

Time

(Minutes)

Air Flow (L/min)

During Exposure

Oxygen Concentration (%)

During Exposure

-3

50

-

0

50

20.8

30

50

-

60

50

-

90

50

-

120

50

20.8

150

50

-

180

50

-

210

50

-

240

50

20.8

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 18.3 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Reaction Products of C4 alcohols and C4 alkenes, in the HsdHanTM : WIST strain rat, was greater than 18.3 mg/L.
Executive summary:

Introduction. A study was performed to assess the acute inhalation toxicity of the test item. The method used followed that described in the OECD Guidelines for Testing of Chemicals (2009) No. 403 “Acute Inhalation Toxicity” and was designed to comply with Method B2 (Inhalation) of Commission Regulation (EC) No. 440/2008, with the exception that only six animals (three males and three females) were utilized during the “limit test”.

Methods. A group of six HsdHanTM: WIST strain rats (three males and three females) was exposed to a vapour atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period.

Results. The mean achieved atmosphere concentration was as follows:

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

18.3

1.36

20.7

The mortality data were summarised as follows:

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

Male

Female

Total

18.3

0/3

0/3

0/6

Clinical Observations. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered quickly to appear normal from Day 2 post-exposure.

Bodyweight. All animals exhibited a slight bodyweight loss during Days 0 to 1 post-exposure, with the exception of one female animal which exhibited a further bodyweight loss during the second week post-exposure, normal bodyweight development was noted during the study.

Necropsy. No macroscopic abnormalities were detected amongst animals at necropsy.

Conclusion. No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 18.3 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Reaction Products of C4 alcohols and C4 alkenes, in the HsdHanTM: WIST strain rat, was greater than 18.3 mg/L.